Summary: During B cell development, antibody gene segments undergo multiple recombinations, first at the heavy chain loci followed by recombination at the light chain loci, to produce surface IgM. Throughout B cell development the expression of surface IgM and other markers is modulated and the pattern of expression of these molecules can identify different stages of differentiation or the activation of B cells. We have studied the contribution of individual germline genes to the pre-immune repertoire and the diversity of the immune response to tetanus toxoid. Our previous studies examined the 3-member Vk10 family as a paradigm for genes that are both expressed against a wide variety of antigens in diverse genetic backgrounds as well as functional V genes that recombine but are not expressed in the mature B cell repertoire. We have shown that for the unexpressed Vk10C gene, there is a selective loss of productive recombinations as the B cell passes from the preB cell stage through the immature B cell in the bone marrow to the mature B cell in the spleen. We have now expanded these findings to include 3 additional gene families with unexpressed functional genes. In addition, more productive recombinations of the unexpressed genes are detected in RS recombination products (a recombination event which inactivates the k locus) whereas non-productive rearrangements of their expressed counterparts are detected in RS-recombination products. Twenty of the 141 functional Vk genes may not be expressed in the pre-immune adult repertoire. Other studies have demonstrated that many functional human Vk genes are also not expressed. The reasons for the lack of expression of such functional genes may be due to negative selection, the inability of the functional gene to pair with the heavy chain, or to insufficient transcription of the Vk gene. Preliminary data of Vk10C suggests that there may be 2-fold less mRNA in preB cells compared to the expressed Vk10A gene but whether or not this difference is significant needs to be examined further. Our studies demonstrating an unequal usage of functional V genes led us to question whether monoclonal antibodies produced by novel technologies such as phage display libraries could use unusual VH-VL pairs. Phage display technology has a vast potential to generate human antibodies against even poorly immunogenic tumor antigens. However, such mAbs do not undergo the normal in vivo positive/negative selection process and could select mAbs utilizing unusual VH-VL combinations which could be immunogenic and/or autospecific. To examine this, we are generating phage display libraries from mice and will compare the repertoire of a phage display anti-tetanus toxoid library to that of a hybridoma library. Phage display libraries will be generated from both unimmunized and immunized mice to mimic the types of phage display libraries utilized for the production of therapeutic mAbs. In addition, our anti-tetanus mAbs will be used to establish an in vitro correlate of protection. Such assays are crucial to development of vaccines for bioterrorism agents and tradtional efficacy trials are unethical and cannot be done.